Complement mediated inhibition of immune precipitation in rheumatoid arthritis: studies on interaction of heat aggregated IgG with IgM rheumatoid factor.

Serum samples from patients with seropositive rheumatoid arthritis contain an inhibitor of complement mediated inhibition of immune precipitation (CMIP). This inhibitory effect can be produced by the addition of either purified monoclonal or polyclonal IgM rheumatoid factor (RF) to human serum. The specificity of the rheumatoid factor influences the degree of inhibition, and when precipitation occurs the rheumatoid factor coprecipitates with the antigen-antibody complex. In rheumatoid sera there was a significant positive correlation between IgM RF concentration and inhibitory activity, though the range of inhibitory activity seen for the same concentration of rheumatoid factor was considerable. Small quantities of heat aggregated IgG (HAGG) had a much greater effect on the measurement in an enzyme linked immunosorbent assay (ELISA) of IgM RF than they did on the inhibitory activity of IgM RF in the CMIP assay. Larger quantities of HAGG initiated complement activation and increased the precipitation of immune complexes. IgM RF reduced the complement activating properties of HAGG by reducing the amount of Clq which bound to the aggregate. The mechanisms by which IgM RF overcomes CMIP in rheumatoid sera may involve its inhibitory effects on the binding of Cl to the antigen-antibody complex.     

Complement-activating properties of IgM rheumatoid factors reacting with IgG subclasses

Summary To estimate the complement-activating property (CAP) of IgM rheumatoid factor (RF), which was purified from synovial fluids of patients with rheumatoid arthritis, in a reaction with each IgG subclass, the activation and binding of C4 in the classical pathway of complement by IgM RF was measured in an enzyme-linked immunosorbent assay using biotinylated F(ab')₂ antibody to human C4. The CAP of IgM RF reacting with IgG3 was significantly higher than that of IgM RFs bound to the other IgG subclasses (P<0.01). These results suggest that IgM RF reacting with IgG3 in synovial fluid could induce a greater degree of complement-dependent inflammation in RA synovium than IgM RF reacting with other IgG subclasses.     

Complement activating properties of monoreactive and polyreactive IgM rheumatoid factors.

OBJECTIVES--To estimate the complement activating properties of monoclonal, monoreactive, and polyreactive IgM rheumatoid factors derived from Epstein-Barr virus transformed B cells isolated from peripheral blood and synovial tissue of patients with rheumatoid arthritis (RA). METHODS--An enzyme linked immunosorbent assay (ELISA) was used to measure the activation of the classical pathway of complement by monoclonal IgM rheumatoid factor. Monoclonal IgM rheumatoid factor was bound to IgG Fc adsorbed onto microtitre plates and then reacted with diluted normal human serum as a source of complement. The activation and binding of C4 were measured with F(ab')2 antibody to human C4. The complement activating property of IgM rheumatoid factor bound to IgG Fc was tentatively expressed as the ratio of the amount of bound C4 to the amount of bound IgM rheumatoid factor. RESULTS--The complement activating property of monoreactive IgM rheumatoid factor was shown to be about three times higher than that of polyreactive IgM rheumatoid factor. CONCLUSIONS--Monoreactive IgM rheumatoid factor with the higher complement activating property would result in a greater degree of complement dependent inflammation and might have a more important pathogenic role in RA than polyreactive IgM rheumatoid factor.     

Isolation and analysis of complement activating aggregates from synovial fluid of patients with rheumatoid arthritis using monoclonal anti-C3d antibodies.

The complement activating aggregates in synovial fluids of patients with rheumatoid arthritis (RA) have been isolated using monoclonal IgM anti-C3d antibodies attached to solid phases, and the content of the material bound has been analysed. High levels of aggregated IgG bearing C3d were found in RA synovial fluids, and IgG was the major immunoglobulin bound from such synovial fluids by anti-C3d Sepharose. A strong correlation was shown between levels of aggregated IgG bearing C3d and complement activation, as judged by C3d levels. Significant (but less strong) relationships were also observed between C3d levels and both complement consuming and C1q binding activity. C3d levels and levels of aggregated IgG bearing C3d were both significantly associated with the numbers of polymorphonuclear leucocytes (PMNs) found in RA synovial fluids. From these results it is concluded that the aggregated immunoglobulins bearing C3d (particularly IgG) isolated from RA synovial fluids are responsible for activating complement and attracting PMNs into the joint space. Radioimmunoassay showed no correlation, however, between levels of aggregated IgG (or IgM) bearing C3d and rheumatoid factor (RF) activity bound by anti-C3d. In addition, the material bound by anti-C3d Sepharose from most synovial fluid polyethylene glycol precipitates did not contain either IgM or IgG RF. Thus both techniques show that the majority of complexes bearing C3d do not contain RF. As the complement fixing aggregates apparently contain only immunoglobulin and complement components the results raise the problem of how the aggregates are formed. It is suggested that RA IgG may remain aggregated after either antigen or antibody (RF) has dissociated from the complex.     

Complement-activating properties of immune complexes are suppressed by IgM rheumatoid factor and enhanced by IgG rheumatoid factor

Summary The effect of rheumatoid factor (RF) on complement-activating capacity of aggregated IgG was investigated. The degree of complement activation induced by the addition of specific amounts of aggregated IgG to patients' sera and normal sera was demonstrated by the inhibition of hemolytic activity (%IHA). The %IHA was significantly lower in rheumatoid arthritis (RA) sera and higher in systemic lupus erythematosus (SLE) sera, compared with normal sera. There was a negative correlation between %IHA and IgMRF/IgGRF ratio in RA and SLE sera, and RA synovial fluid. The %IHA and IgGRF were positively correlated in RA sera. The IgMRF/IgGRF ratio was significantly lower in SLE sera than in RA sera and systemic sclerosis sera, and was significantly lower in RA synovial fluid than in osteoarthritis synovial fluid.Isolated RF, consisting of mostly IgMRF class, inhibited complement-activating properties of aggregated IgG, depending on the concentration of RF. Isolated RF was further purified by the fractionation using high pressure liquid chromatography, and IgGRF and IgMRF were obtained. IgMRF significantly suppressed the complement-activating capacity of aggregated IgG, whereas IgGRF promoted it. These observations suggest that IgMRF acts protectively, while IgGRF induces inflammation.Thus, the expression of the biological activity of RF with special reference to immune complex interaction mainly depends on the IgMRF/IgGRF ratio.     

HUMAN COMPLEMENT ACTIVATION BY SELF-ASSOCIATED IgG RHEUMATOID FACTORS

IgG rheumatoid factors (IgG–RFs) undergo concentration-dependent self-association into dimers and higher polymers, as previously reported. The interactions of purified IgG–RF from the plasma of 3 patients with rheumatoid arthritis, with guinea pig and human complement were studied. Self-associating IgG–RFs were isolated by affinity columns and gel filtration. These preparations contained no detectable IgM and were composed only of IgG subclasses known to fix complement. Complement utilization of IgG–RF was compared with that of monomeric IgG, heat-aggregated IgG, and soluble rabbit IgG immune complexes. Although incubation of IgG–RF or monomeric IgG with 3 units of guinea pig or human complement resulted in decreased hemolysis of sheep erythrocytes sensitized with IgM hemolysin, these substances were less than 100 times as effective as heat-aggregated IgG or soluble immune complexes. The ability of human or guinea pig complement that had been incubated with IgG–RF to restore hemolytic activity to C4-deficient guinea pig serum served to distinguish Clq binding from complement cascade activation. IgG–RFs and monomeric IgG did not activate guinea pig complement cascade in contrast to aggregated IgG. IgG–RFs, however, activated human complement cascade; monomeric IgG only bound human Clq. These results indicate that self-associated IgG–RFs can activate human complement in fluid phase, but less effectively than aggregated IgG or large-latticed immune complexes.     

C1q-bearing immune complexes detected by a monoclonal antibody to human C1q in rheumatoid arthritis sera and synovial fluids

Summary Using a monoclonal antibody directed against the C-chain of human C1q, we detected C1q-bearing immune complexes (IC) in sera and synovial fluids of rheumatoid arthritis (RA) patients. In a sandwich-ELISA, C1q-bearing IC were captured by the solid-phase monoclonal antibody and then detected with peroxidase-labeled F(ab)₂-antibodies to either human IgG or IgM. The results of this assay were compared to an ELISA-modification of the C1q-solid-phase binding assay (C1q-SPBA). C1q-bearing IC were detected in 81.1% of RA-sera and the 65.2% of RA-synovial fluids. IgG as well as IgM was present in 72.6% of the sera and 70% of the synovial fluids which were positive in both assays. Most RA sera that were only positive for C1q-bearing IC, contained IgG alone (81.5%). The corresponding synovial fluids showed IgG alone (53%) or both IgG and IgM (41.1%). IgM alone (25%) could be detected in sera, e.g. in juvenile forms of RA. The levels of IC were higher in synovial fluid than in paired serum. In comparison to normal human serum (NHS) and patients with osteoarthritis, complement activity (CH50 titers) and C1q-values in patients with RA were frequently elevated. Since the formation of C1q-bearing IC is an indicator for the classical complement pathway activation, an assay with monoclonal anti-C1q antibody may be a useful tool in the diagnosis of rheumatoid diseases.     

Serum IgG and IgM rheumatoid factors and complement activation in extraarticular rheumatoid disease

Rheumatoid factors (RF) may participate in both synovial and extraarticular (EA) inflammation in rheumatoid disease (RD). The relative roles of serum IgG RF and IgM RF in extraarticular rheumatoid disease (EARD) are unclear, as is the importance of complement (C) activation by these proteins. To investigate the relation of C activating properties of IgM RF (RF CAP) and total IgG RF and IgM RF to EARD we compared 18 patients with only articular disease to 27 patients with various EA manifestations (nodules, cutaneous vasculitis, neuropathy, Felty's syndrome) using radioimmunoassays for IgG and IgM RF and an established hemolytic assay for C activation by IgM RF. We calculated RF CAP by determining the mean hemolysis of sensitized SRBC/ml of RF serum (MH/ml). Normal volunteers and patients with other inflammatory arthritides served as controls. Controls had negligible amounts of IgG RF, IgM RF, and RF CAP. Mean IgG and IgM RF levels and RF CAP values were significantly higher in patients with EARD than those in the arthritis-only (AO) group. Mean IgM RF concentrations and IgM RF CAP correlated with each other and EARD. IgG RF also correlated with IgM RF and EARD, but did not contribute to RF CAP or EARD when adjusted for IgM RF. Further, some patients had high RF CAP values despite modest IgM RF levels. These data suggest that quantitative differences in IgM RF CAP and total IgM RF may be more important than IgG RF as determinants of EARD.     

Relationship of serum IgG rheumatoid factor to IgM rheumatoid factor and disease activity in rheumatoid arthritis

Rheumatoid factors (RF) constitute the major autoantibodies in rheumatoid arthritis (RA). RF are directed against IgG Fc, are polyclonal, and are predominantly of the IgG and IgM classes. RF may participate in both synovial and extraarticular inflammation in RA, although the precise roles of serum IgG and IgM RF are unclear. The purpose of our study was to correlate serum IgG RF with serum IgM RF levels measured by radioimmunoassay and with clinical disease activity in 42 prospectively evaluated seropositive RA patients. IgM RF correlated with IgG RF levels and articular disease activity. IgG RF correlated with IgM RF but not with articular disease activity when adjusted for IgM RF.     

Serum and synovial fluid IgG,IgA and IgM antigammaglobulins in rheumatoid arthritis

Antigammaglobulins of IgG, IgA and IgM classes were measured in normal individuals and in patients with osteoarthritis or rheumatoid arthritis. Serum IgG and IgA and synovial fluid IgG antigammaglobulin levels were significantly higher in patients with rheumatoid arthritis than in other individuals, with highest levels occurring in patients with positive latex fixation tests. IgM antigammaglobulins were elevated only in patients with latex positive rheumatoid arthritis. Increased serum levels of IgG, IgA and IgM antigammaglobulins were each associated with clinical findings of severe rheumatoid arthritis. Increased levels of serum and synovial fluid IgG and IgM antigammaglobulins were each associated with diminished serum and synovial fluid complement levels.     

Immunoprecipitates used as a reagent for rheumatoid factors

IgM and IgG rheumatoid factors (RFs) were purified by affinity chromatography and gel filtration. The preparations thus obtained were used as standards in a radio-immunoassay for RF detection. In this assay, RFs were reacted with immunoprecipitates, and the RFs were detected with radiolabelled (F(ab')2 fragments specific for human IgM or IgG. The reproducibility of the assay was higher when RF content was expressed relative to the standards and not directly relative to tracer binding. It was found that the presence of IgM RF did not affect the measurement of IgG RF in this RIA, since the addition of neither mono- nor polyclonal IgM RF to a donor serum resulted in increased IgG RF measurements. 100 sera were analysed and were consistently positive in only one of the tests: sheep cell agglutination or latex fixation. The 75% of the sera which were positive only in the latex fixation test were positive for IgM RF in the radio-immunoassay, indicating that RFs in this type of serum were not specific solely for human IgG.     

Rheumatoid factor isotypes and circulating immune complexes in rheumatoid arthritis

Solid phase enzyme immunoassays were here used to quantify rheumatoid factors (RF) of the IgM, IgG and IgA classes and the immune complexes (IK) by their ability to bind to C1q or conglutinin in both the serum and synovial fluid of patients with rheumatoid arthritis (RA). Elevated serum levels of any RF isotype could be found in all patients with seropositive RA (IgM: 63%, IgG: 87%, IgA: 90%). Seronegative patients with RA presented to a significantly lesser extent with elevated levels of all the RF isotypes tested (IgM: 0%, IgG: 40%, IgA: 32%). Synovial fluid RF levels were significantly higher in SPRA patients than in SNRA patients with the exception of IgG-RF. All of the RF classes in both RA groups, however, were elevated when compared to RF in the synovial fluid of patients with osteoarthrosis. Both C1q binding and conglutinin binding immune complexes were significantly higher in the synovial fluid than in the serum of RA patients. The erythrocyte sedimentation rate and plasma iron levels were correlated with the levels of C1q binding immune complexes (IC) in the synovial fluid; total iron binding capacity showed an inverse relationship to synovial fluid IgG-RF levels. A radiographic index was also correlated with IgG-RF levels in the synovial fluid. Extraarticular manifestations were significantly more frequent in patients with elevated serum levels of IgM-RF or conglutinin binding IC. These findings indicate that IgG-RF in the synovial fluid and the formation of IC determined by their ability to bind C1q seem to be closely related to clinical features of local disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restricted heterogeneity of polyclonal rheumatoid factors

The electrophoretic pattern of rheumatoid factor (RF) was investigated in 40 polyclonal sera, by using radiolabeled IgG. Thirty sera specifically bound IgG aggregates, correlating with their RF titer. The binding pattern was monoclonal or oligoclonal. The molecules responsible were classic RF antibodies, as shown by using purified IgM, inhibition experiments, and by the optimal size of aggregates (2,000-4,000 kd). These data show that RF heterogeneity is restricted in polyclonal sera, and this can have a bearing on several mechanisms.     

Direct activation of neutrophil chemiluminescence by rheumatoid sera and synovial fluid.

The majority of paired sera and synovial fluids from 21 patients with rheumatoid arthritis produced a rapid chemiluminescent response when incubated with human neutrophils. Synovial fluid gave considerably higher responses than the paired serum specimen. In contrast little or no response was found with paired sera and joint fluid taken from patients with gout, psoriasis, and osteoarthritis and with sera from healthy donors. A similar chemiluminescent response was observed when neutrophils were preincubated with large aggregates of heated human gammaglobulin (HAGG), which were used as a model of immune complexes. Smaller nonreactive aggregates of gammaglobulin became reactive after preincubation with a purified monoclonal rheumatoid factor (mRF) which had a high avidity for aggregated IgG. The addition of this monoclonal rheumatoid factor also caused enhancement of chemiluminescence by rheumatoid sera. Further evidence suggesting that the active material found in these rheumatoid specimens contained complexed immunoglobulin was obtained by indirect immunofluorescence. Neutrophils developed intracellular immunoglobulin inclusions after preincubation in reactive rheumatoid sera but not with nonreactive or normal sera. However, activation of neutrophil chemiluminescence by rheumatoid specimens did not correlate significantly with levels of rheumatoid factor or immune complexes suggesting that the activating complexes were of a particular type. In conclusion we have shown the direct activation of neutrophil chemiluminescence by rheumatoid sera synovial fluid and suggest that the activation is caused by large IgG-containing immune complexes. It is possible that this activation may have important implications in the immunopathogenesis of the rheumatoid inflammatory process.     

Detection of low molecular weight IgM by immunoblot analysis in rheumatoid arthritis

The immunoblot technique was used to detect low molecular weight IgM (LMWIgM) in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA). LMWIgM was detected in 64% of 58 RA sera and in 47% of 17 RA SF. The levels of IgM and rheumatoid factor (RF) were significantly higher in the positive sera of LMWIgM. Sequential studies revealed that LMWIgM appeared in the serum while the titer of RF was high. Our analysis also suggested the presence of several other oligomeric LMWIgM with monomeric IgM.     

A sensitive radioimmunoassay for quantitation of igm rheumatoid factor

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor (RF) in biologic fluids has been developed. Binding curves for monoclonal IgM RF and polyclonal IgM rheumatoid factors were similar under the conditions utilized for the assay. Human IgG did not interfere with the detection of IgM RF by this method. Small quantities (≤ 0.2%) of nonspecific binding by nonRF IgM to the human IgG coated tubes utilized in the assay were corrected for by assaying samples in parallel bovine serum albumin coated control tubes. As expected, patients with seropositive rheumatoid arthritis (RA) had significantly higher concentrations of IgM RF than seronegative RA patients (mean ± 1 SD = 652 ± 553 μg/ml versus 11.3 ± 13.3 μg/ml, P < 0.001). In contrast, all normal control sera assayed to date contained < 0.1 μg/ml of IgM RF. The capacity of the assay to detect nanogram quantities of IgM RF should permit investigation of the cellular mechanisms underlying RF production.     

Antigammaglobulin (rheumatoid factor) activity of human IgG subclasses.

A sensitive radioimmunoassay has been applied to the study of rheumatoid factor activity of IgM and IgG subclasses in serum and synovial fluid of patients with rheumatoid arthritis. Although rheumatoid factor activity was found in all four subclasses, its distribution was not the same as that of the total subclasses in normal sera. Results are discussed in relation to the possible clinical value of measuring IgG rheumatoid factor in sera and synovial fluids of patients with rheumatoid arthritis.     

Enzyme immunoassay of complement-binding rheumatoid factors

Summary We describe an enzyme immunoassay for the determination of complement-binding rheumatoid factors. Polystyrene tubes are coated with heat aggregated human IgG. The rheumatoid factors (RFs) of patients heat inactivated sera are allowed to bind to aggregated IgG and thereafter saturated with fresh human complement. The amount of C 3 complement bound is measured by indirect enzyme immunoassay. The levels of complement binding RFs were measured in 30 patients with seropositive rheumatoid arthritis (RA), in 19 patients with systemic lupus erythematosus (SLE), and in 30 healthy control subjects. Compared to the controls high levels of complement-binding RFs were found both in RA and in SLE (P<0.0005). The mean level of the complement binding RFs was higher (P<0.05) in active than in inactive SLE. Even though the 19 S IgM RF bound complement, in RA no correlation was found between the level of complement binding RFs and Waaler-Rose titre, but the level of complement binding RF correlated with the levels of nonagglutinating IgM RF (r=0.56, P<0.01) and IgG RF (r=0.70, P<0.001) that were obtained by enzyme immunoassay.     

Monoclonal antibody 6b6.6 defines a cross-reactive kappa light chain idiotope on human monoclonal and polyclonal rheumatoid factors

Mouse monoclonal antibody (MAb) 6B6.6 was raised against a cross-reactive idiotope (CRI) present on the light chains of 2 human IgM paraproteins with rheumatoid factor (RF) activity. The MAb inhibited the IgG-binding activity of these proteins, and thus appears to react with an epitope located at or near the RF-binding site. Enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting studies indicate that the 6B6.6 CRI is associated with kIIIa sub-subgroup light chains, is not related to the Wa, Po, and Bla RF cross-idiotypic specificities, and is clearly distinct from the kIIIb-associated CRI detected by MAb 17.109. Using an ELISA, we detected 6B6.6 CRI in 59% of 107 sera and 48% of 50 synovial fluids from patients with seropositive rheumatoid arthritis (RA). However, the quantities of CRI-positive RF were small, and the amount of CRI-positive RF did not correlate with the amount of IgM-RF. The 6B6.6 CRI was shown to occur primarily in the IgM fraction of RA sera by both chromatographic studies and isotype-specific ELISA, although small quantities appeared to be associated with IgA and IgG in some sera. The presence of 6B6.6 CRI on both monoclonal and polyclonal RF is consistent with the view that both are derived, at least in part, from a common gene pool. However, its occurrence in relatively low levels suggests that the number of germline genes encoding for RF is large or that extensive mutation occurs in the course of RF expression in RA.     

Absence of antibody to cyclic citrullinated peptide in sera of non-arthritic patients with chronic hepatitis B virus infection

The objective of this study was to investigate if antibody to cyclic citrullinated peptide (anti-CCP) is detected in sera of patients with chronic hepatitis B virus (HBV) infection. Serum anti-CCP and IgA, IgG, and IgM rheumatoid factor (RF) isotypes were measured by enzyme-linked immunosorbent assay on 176 non-arthritic patients with HBV infection. IgA RF, IgG RF, and IgM RF were detectable in 29.5, 21, and 18.8% of the tested sera, respectively, with a total seropositivity rate of 42.7%. Marginally elevated anti-CCP was detected in one patient (0.6%). By regression analysis, there was no statistically significant association between the serum levels of anti-CCP and serum IgA, IgG, or IgM RF (R ² = 0.033, with respective p values of 0.224, 0.297, and 0.334). In conclusion, anti-CCP was rarely detected in non-arthritic patients with HBV infection in contrast to RF. Thus, testing for anti-CCP may be a useful tool for the diagnosis of rheumatoid arthritis in this population.